Yearly influenza epidemics affect about 5 - 15% of the world's population and estimates of annual mortality range from 250,000 - 500,000, including approximately 30,000 deaths and 200,000 hospitalizations in the United States. In addition, the likelihood of a severe pandemic caused by a newly emergent strain of influenza virus is very high, given that three such pandemics were recorded in the 20th century, plus the swine H1N1 pandemic of 2009. The most devastating of these, the 1918 Spanish influenza, was associated with an estimated 40 million deaths worldwide. The annual vaccine for seasonal influenza is only partially effective in prevention of disease. Likewise, currently available anti-influenza drugs such as amantadine and oseltamivir are only partially effective in treatment prophylaxis, and also suffer from problems of drug resistance. Therefore, there is an urgent need for additional anti-influenza therapeutics that target unexploited aspects of viral biology. The proposed studies are directed at developing new drugs that can combat influenza virus. The viral NS1 protein is an attractive drug target because it is essential for virus replication in vivo. Small molecules that inhibit NS1 function are expected to block virus replication, and hence disease. During Phase I, two chemical series were developed that specifically inhibit NS1 function during infection. These compounds inhibit virus replication in cell culture and an animal model. The goal for Phase II is to develop these series to be ready for IND-enabling pharmacology and toxicology in Phase III. In Aim 1, medicinal chemistry approaches will be used to create highly potent, non-toxic analogs. In Aim 2, purified NS1 protein will be used in biochemical assays to determine binding constants for the newly synthesized analogs, and X-ray crystallography will be employed to understand the structure of ligand-drug complexes. These methods will support the design of better NS1 inhibitors. In Aim 3, the analogs will be assessed according to several criteria including antiviral activity, cytotoxicity, induction of cellular interferon, and in vitro ADME properties. Aim 4 will culminate with in vivo testing in BALB/c mice, including MTD, PK and efficacy studies.